Biology subjects

Brownstone, R. M.

Publications and source records attributed to Brownstone, R. M..

15 recordsLinked to original sources

bioRxiv2026

Spinal circuits encode a map of the trunk to control skin twitches

Reflexive skin twitches are stereotypical behaviours, triggered in most mammals by mechanosensory stimuli. The neural circuits controlling this behaviour are thought to lie in the spinal cord, with motor neurons positioned in the lower cervical spinal cord innervating the cutaneous maximus muscle responsible for the twitches. The spatial matching of the motor output to the location of the sensory stimulus points to selective innervation of particular cutaneous maximus motor neurons by specific sensory-responsive circuits organised in a spatially-dependent manner. Using mouse genetics and viral tracing, we observed that a subset of dorso-lateral dI3 neurons forms ascending projections to motor neurons mediating skin twitches. Their projections are somatotopically organized to map a two dimensional space onto specific sub-compartments of the cutaneous maximus motor pool. Furthermore, direct optogenetic stimulation of thoraco-lumbar dI3 ascending projections in the cervical spinal cord induces skin twitches. Together, we demonstrate the circuit basis of a spinal sensory-motor representation of the dorsolateral trunk that participates in an ethological behaviour shared by most mammals allowing them to reduce the burdens of irritants such as insects.

neuroscience↗

bioRxiv2026

In vivo motor unit decoding and in vitro cellular characterisation of spinal circuits for urination in adult mice

Urinary dysfunction affects billions of individuals worldwide; however, the fundamental cellular and circuit properties that govern perineal motor control remain largely unknown, serving as a functional "black box". Here, we describe several methods that, when used in concert, characterise cellular, synaptic, and motor unit properties underlying the control of urination in adult mice. High-density electromyography combined with real-time cystometry were used to study external urethral sphincter (EUS) motor units, which follow a hierarchical ("onion skin") recruitment pattern during bladder filling. The transition to the voiding phase is marked by inhibition, followed by synchronised bursts. Furthermore, through concurrent recordings of ischiocavernosus (IC) muscles, the relationship between IC and EUS motor units could be studied to look for shared common inputs that could shed light on circuitry. Whole-cell patch-clamp recordings from retrogradely identified neurons revealed a fundamental biophysical divergence: urinary parasympathetic preganglionic neurons (PPGN) are significantly smaller and more excitable than somatic EUS and IC motoneurons and lack the recurrent excitatory and inhibitory circuits present in both EUS and IC motor pools. Finally, using a novel pressure-clamp preparation, we showed that acute tibial nerve stimulation (a widely used treatment for urinary dysfunction) evokes short-latency inhibition of EUS motor units. Collectively, these methods can be used to delineate patterns of motor unit recruitment, local recurrent microcircuit architecture, and distinct biophysical properties of the perineal motor system, providing mechanistic insights into urinary function.

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bioRxiv2025

Evidence of spinal cord comparator modules for rapid corrections of movements

Successful movement requires continuous adjustments in response to changes in internal and external environments. To do so, neural circuits continuously compare efference copies of motor commands with sensory feedback to respond to sensory prediction errors. Some responses need to be very fast, indicating that they likely occur within spinal cord circuits. Here, we describe spinal cord circuits involving dI3 neurons, showing that they receive multimodal sensory inputs and direct efferent copies from both Renshaw cells and motor neurons. We further show that they form connections to motor pools. Reducing dI3 neuronal activity diminished stumbling responses, as did disrupting Renshaw cell circuits, providing evidence for a comparator role of dI3 neurons for online corrections. Together, our findings reveal a pivotal role for dI3 neurons functioning as comparators of efference copies and external sensory feedback to mediate rapid corrections of ongoing movements.

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bioRxiv2025

Ethologically relevant behavioural assay for investigating reach and grasp kinematics during whole-body motor control in mice

Reach and grasp are critical components of skilled mammalian motor control and their detailed analysis in rodents has been key to deepening our understanding of prehension in the context of health and disease. However, most studies investigating these behaviours focus on isolating forelimb movements with little regard to the whole-body movements that are key for effective behaviour. To address this issue, we designed a novel behavioural approach to investigate reach and grasp during whole-body, vertical locomotion in mice. Using a customizable transparent climbing surface, we show that our behavioural approach can extract key kinematic features of climbing. Mouse climbing gait reflects aspects of quadrupedal locomotion, showing similar phase dependencies on increasing speed, including reduced stance (i.e. grasp) time and duty factor. Analysis of multi-limb coordination indicated that climbing revolves around anti-phasic forepaw movements with less consistency in interlimb coordination in the hindpaws. Fore- and hindpaws also differed in their reach trajectories and velocity profiles. The flexibility of this approach also allows for tailored climbing configurations, which we use to show that mice can adapt to and overcome vertical obstacles. By leveraging naturalistic climbing, our modular behavioural approach enables investigation of complex prehensile behaviours and facilitates new study into the neural circuits underlying whole-body skilled motor control.

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bioRxiv2025

Personalized mapping of inhibitory spinal circuits via neural decoding of high-density electromyography and in silico modelling

Studying human motoneuron activity through electromyography (EMG) can yield insights into the operation of fundamental spinal cord microcircuits. Traditional surface and needle electromyography (EMG) methodologies have limited capacity to shed light on the diversity of motor unit (MU) control strategies that may be unique to each individual. Here, we employed high-density surface EMG (HDsEMG) to sample multiple MUs per subject to investigate the dynamics of inhibitory spinal microcircuits in both upper and lower limb control. We characterised the net inhibition as a function of individual MU firing rates, revealing subject-specific relationships. In silico modelling replicated these experimental characteristics and suggested that properties of the inhibitory currents rather than motoneuron size are responsible for net functional inhibition. Our results show that HDsEMG can highlight distinct control strategies across circuits and motor pools, revealing subject-specific properties of inhibitory spinal microcircuits. TEASERHigh-density surface EMG electrodes can reveal the functional properties of inhibitory spinal circuits

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bioRxiv2024

Spinal microcircuits go through multiphasic homeostatic compensations in a mouse model of motoneuron degeneration

In many neurological conditions, early-stage neural circuit adaption can preserve relatively normal behaviour. In some diseases, spinal motoneurons progressively degenerate yet movement is initially preserved. We therefore investigated whether these neurons and associated microcircuits adapt in a mouse model of progressive motoneuron degeneration. Using a combination of in vitro and in vivo electrophysiology and super-resolution microscopy, we found that, early in the disease, neurotransmission in a key pre-motor circuit, the recurrent inhibition mediated by Renshaw cells, is reduced by half due to impaired quantal size associated with decreased glycine receptor density. This impairment is specific, and not a widespread feature of spinal inhibitory circuits. Furthermore, it recovers at later stages of disease. Additionally, an increased probability of release from proprioceptive afferents leads to increased monosynaptic excitation of motoneurons. We reveal that in motoneuron degenerative conditions, spinal microcircuits undergo specific multiphasic homeostatic compensations that may contribute to preservation of force output.

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bioRxiv2023

On the origin of F-wave: involvement of central synaptic mechanisms

Neurophysiological methods are used widely to gain information about motoneuron excitability and axon conduction in neurodegenerative diseases. The F-wave is a common biomarker used to test motoneuron properties in the diagnosis of neurological diseases. Although the origin of the F-wave is a subject of debate, the most widely accepted mechanism posits that the F-wave is generated by the backfiring of motoneurons stimulated antidromically from the periphery. In this study, we developed an ex vivo mouse sciatic nerve-attached spinal cord preparation with sensory axons severed in which stimulation of the whole sciatic nerve or its tibial branch evoked responses with the electrophysiological signatures of F-waves. Manipulations of synaptic transmission by either removal of extracellular calcium or block of post-synaptic glutamate receptors abolished these responses. These results suggest that F-waves are mediated by spinal microcircuits activated by recurrent motor axon collaterals via glutamatergic synapses.

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bioRxiv2022

Kv2 conductances are not required for C-bouton mediated enhancement of motoneuron output

Neural motor systems have evolved complex circuits that afford animals a range of behaviours essential for survival. C-bouton synapses arising from cholinergic V0C interneurons amplify motoneuron activity via muscarine type 2 receptors, thus increasing muscle contraction force. Recent work in neonatal mouse motoneurons suggests that delayed rectifier currents carried by post-synaptically clustered KV2.1 channels are crucial to C-bouton amplification. Here we use a motoneuron conditional KV2.1 knockout to show that while KV2.1 modulates maximal firing in neonatal mice, its removal minimally affects either mature motoneuron firing or the enhanced firing rates in response to exogenously applied muscarine. In keeping with this, pharmacological blockade of KV2 currents has minimal electrophysiological effects on mature motoneurons. Furthermore, amplification of electromyography activity during high force tasks was unchanged following KV2.1 deletion. We next show that KV2.2 is also expressed by spinal motoneurons and colocalises with KV2.1 opposite C-boutons. We suggest that the primary function of KV2 proteins - KV2.1 and KV2.2 - is non-conducting in motoneurons, and that KV2.2 can function in the absence of KV2.1, perhaps to ensure the integrity of the synapse.

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bioRxiv2022

Pathophysiology of Dyt1 dystonia is mediated by spinal cord dysfunction

Dystonia, a neurological disorder defined by abnormal postures and disorganised movements, is considered to be a neural circuit disorder with dysfunction arising within and between multiple brain regions. Given that spinal circuits constitute the final pathway for motor control, we sought to determine their contribution to the movement disorder. Focusing on the most common inherited dystonia, DYT1-TOR1A, we confined a conditional knockout of Tor1a to the spinal cord and dorsal root ganglia (DRG) and found that these mice recapitulated the phenotype of the human condition, developing early onset generalised torsional dystonia. Physiologically, these mice bore the hallmark features of dystonia: spontaneous contractions at rest, excessive sustained contractions during voluntary movements including co-contractions of motor antagonists, and altered sensory-motor reflexes. Furthermore, spinal locomotor circuits were impaired. Together, these data challenge current understanding of dystonia, and lead to broader insights into spinal cord function and movement disorder pathophysiology.

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bioRxiv2021

Spinal neurons innervating multiple local and distant motor pools

Motor neurons control muscle contractions, and their recruitment by premotor circuits is tuned to produce accurate motor behaviours. To understand how these circuits coordinate movement across and between joints, it is necessary to understand whether spinal neurons pre-synaptic to motor pools, project to more than one motor neuron population. Here, we used modified rabies virus tracing in mice to investigate premotor INs projecting to synergist flexor or extensor motor neurons, as well as those projecting to antagonist pairs of muscles controlling the ankle joint. We show that similar proportions of premotor neurons diverge to agonist and antagonist motor pools. Divergent premotor neurons were seen throughout the spinal cord, with decreasing numbers but increasing proportion with distance from the hindlimb enlargement. In the cervical cord, divergent long descending propriospinal neurons were found in contralateral lamina VIII, had large somata, were excitatory, projected to both lumbar and cervical motoneurons, and were at least in part of the V0 class. We conclude that distributed spinal premotor neurons coordinate activity across multiple motor pools and that there are spinal neurons mediating co-contraction of antagonist muscles.

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bioRxiv2021

On the distribution of spinal premotor interneurons

Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized we used multiple complementary viral-tracing approaches to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components. The paper can be downloaded in executable format as a MATLAB live script from https://github.com/marcobeato/Spinal_premotor_interneurons_controlling_antagonistic_muscles_are_spatially_intermingled, where all the data are available An R version of the executable paper is available at https://mybinder.org/v2/gh/rronzano/Spinal_premotor_interneurons_controlling_antagonistic_muscles_are_spatially_intermingled.git/HEAD?urlpath=rstudio

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bioRxiv2021

C-bouton components on rat extensor digitorum longus motoneurons are resistant to chronic functional overload

Mammalian motor systems adapt to the demands of their environment. For example, muscle fibre types change in response to increased load or endurance demands. However, for adaptations to be effective, motoneurons must adapt such that their properties match those of the innervated muscle fibres. We used a rat model of chronic functional overload to assess adaptations to both motoneuron size and a key modulatory synapse responsible for amplification of motor output, C-boutons. Overload of Extensor Digitorum Longus (EDL) muscles was induced by removal of their synergists, Tibialis Anterior (TA) muscles. Following 21 days survival, EDL muscles showed an increase in fatigue resistance and a decrease in force output, indicating a shift to a slower phenotype. These changes were reflected by a decrease in motoneuron size. However, C-bouton complexes remained largely unaffected by overload. The C-boutons themselves, quantified by expression of vesicular acetylcholine transporter, were similar in size and density in the control and overload conditions. Expression of the post-synaptic voltage-gated potassium channel (KV2.1) was also unchanged. Small conductance calcium activated potassium channels (SK3) were expressed in most EDL motoneurons, despite this being an almost exclusively fast motor pool. Overload induced a decrease in the proportion of SK3+ cells, however there was no change in density or size of clusters. We propose that reductions in motoneuron size may promote early recruitment of EDL motoneurons, but that C-bouton plasticity is not necessary to increase the force output required in response to muscle overload.

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bioRxiv2021

Elimination of glutamatergic transmission from Hb9 interneurons does not impact treadmill locomotion

The spinal cord contains neural circuits that can produce the rhythm and pattern of locomotor activity. It has previously been postulated that a rhythmogenic population of glutamatergic neurons, termed Hb9 interneurons, contributes to this rhythmogenesis. The homeobox gene, Hb9, is expressed in these interneurons as well as motor neurons. We developed a mouse line in which cre recombinase activity is inducible in neurons expressing Hb9. We then used this line to eliminate vesicular glutamate transporter 2 from Hb9 interneurons, and found that there were no deficits in treadmill locomotion. We conclude that glutamatergic neurotransmission by Hb9 interneurons is not required for locomotor rhythmogenesis. The role of these neurons in neural circuits remains elusive.

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bioRxiv2021

Local brainstem circuits contribute to reticulospinal output in the mouse

Glutamatergic reticulospinal neurons in the gigantocellular reticular nucleus (GRN) of the medullary reticular formation can function as command neurons, transmitting motor commands to spinal cord circuits. Recent advances in our understanding of this neuron-dense region have been facilitated by the discovery of expression of the transcriptional regulator, Chx10, in excitatory reticulospinal neurons. Here, we address the capacity of local circuitry in the GRN to contribute to reticulospinal output. We define two sub-populations of Chx10-expressing neurons in this region, based on distinct electrophysiological properties and somata size (small and large), and show that these correspond to local interneurons and reticulospinal neurons, respectively. Using focal release of caged-glutamate combined with patch clamp recordings, we demonstrated that Chx10 neurons form microcircuits in which the Chx10 interneurons project to and facilitate the firing of Chx10 reticulospinal neurons. We discuss the implications of these microcircuits in terms of movement selection. SIGNIFICANCE STATEMENTReticulospinal neurons in the medullary reticular formation play a key role in movement. The transcriptional regulator Chx10 defines a population of glutamatergic neurons in this region, a proportion of which have been shown to be involved in stopping, steering, and modulating locomotion. While it has been shown that these neurons integrate descending inputs, we asked whether local processing also ultimately contributes to reticulospinal outputs. Here, we define Chx10-expressing medullary reticular formation interneurons and reticulospinal neurons, and demonstrate how the former modulate the output of the latter. The results shed light on the internal organization and microcircuit formation of reticular formation neurons.

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bioRxiv2020

Spinal motoneuron firing properties mature from rostral to caudal during post-natal development of the mouse

Altricial mammals are born with immature nervous systems comprised of circuits that do not yet have the neuronal properties and connectivity required to produce future behaviours. During the critical period of post-natal development, neuronal properties are tuned to participate in functional circuits. In rodents, cervical motoneurons are born prior to lumbar motoneurons, and spinal cord development follows a sequential rostro-caudal sequence. Here we asked whether birth order is reflected in the post-natal development of electrophysiological properties. We show that motoneurons of both segments have similar properties at birth and follow the same developmental profile, with maximal firing increasing and excitability decreasing into the 3rd post-natal week. However, these maturative processes occur in cervical prior to lumbar motoneurons, correlating to the timing of arrival of descending systems. These results suggest that motoneuron properties do not mature by cell autonomous mechanisms alone, but rather depend on developing descending and spinal circuits.

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